Nucleic acid-based diagnostic tests are widely used in medicine, forensics and environmental applications. Detecting variations in a particular nucleic acid sequence provides information about polymorphisms and mutations, including disease-causing mutations. For example, detecting an individual's mutant genotype provides disease carrier status for genetic counseling. A more challenging task is detecting somatic mutations that arise in tissues and cause disease or disease progression. For example, many cancers are caused by a particular mutation. Later, additional mutations accumulate in cancer cells during tumor progression. See Lea et al. (2007) Genetic pathways and mutation profiles of human cancers: site and exposure-specific patterns, Carcinogenesis, 28(9):1851-1858. Downward, J. (2003) Targeting RAS signaling pathways in cancer therapy (2005), Nature Rev. Cancer, 3:11-22. These mutations are predictive of disease outcome and of response to therapy. See Ikediobi et al. (2008) Somatic pharmacogenomics in cancer, Pharmacogenomics J., 8:305-314, Pao et al. (2005) KRAS mutations and primary resistance of lung adenocarcinomas to gefitinib and or erlotinib, PLoS Medicine, 2(1), e17. The ability to detect such mutations is extremely useful in cancer diagnostics and treatment. However, detection of the mutations, especially early detection, faces many technical challenges.
A major challenge in detecting a cancer-related mutation is the rare nature of the mutation, especially when it first arises in a single cell during carcinogenesis. Initially, only a subpopulation of cells carries the mutation, while the surrounding cells still carry the wild-type sequence. Therefore, in a nucleic acid isolate, the newly-mutated nucleic acid is obscured by the excess of the wild-type nucleic acid. Many allele-specific detection methods (such as allele-specific PCR) involve preferential amplification of the sequence of interest (mutant sequence) over the undesired sequence (wild-type sequence). Unfortunately, in most cases, the selectivity of the assay is not perfect, i.e. the undesired sequence is also amplified, but a lot less efficiently than the desired sequence. Because the undesired (wild-type) sequence is present in great molar excess over the mutant sequence, the disadvantage is erased and the wild-type sequence is amplified predominantly, obscuring the presence of the mutant sequence.
Some methods have been developed in response to this challenge. For example, U.S. Pat. No. 5,849,497 and application Ser. No. 12/186,311, filed on Aug. 5, 2008, teach using an amplification blocker that would prevent the amplification of the competing undesired sequence. In this approach, the blocker is a non-extendible oligonucleotide which forms a stable hybrid with the undesired sequence (but not with the desired sequence) downstream of one of the amplification primers. When the blocker is stably hybridized, a DNA polymerase deficient in the 5′-3′-nuclease activity is unable to complete the extension of the primer. The success of this approach depends on the sequence divergence between the desired and the undesired sequences. The approach works best where there are multiple differences between the sequences, ensuring that the hybrid between the blocker and the sequence to be suppressed is stable, while the hybrid between the blocker and the sequence to be amplified is unstable.
The above method has several technical limitations. A longer blocker oligonucleotide is more efficient at blocking, but may be unable to discriminate, thus blocking amplification of all sequence variants. A shorter blocker may be unable to block any amplification efficiently. In some sequence contexts, there may be so few differences that a blocker is capable of very weak discrimination. Therefore, in some loci of clinical interest, the blocker alone is insufficient to solve the technical problems of allele-specific amplification.